The objective of the proposed research is to examine the properties of several folate metabolizing enzymes derived from human cells in culture, with emphasis on the structure of folate analogs as it relates to their substrate and inhibitor activity. The enzymes to be included in the study are TMP synthetase (TS), H2PteGlu reductase (D FR), and the 5, 10 CH2H4PteGlu De-hydrogenase - 5, 10 CH equals H4PteGlu Cyclohydrolase -l0 CHO H4PteGlu Synthetase Complex (DCS). We have previously purified by procedures developed in our laboratory and characterized TS from AML peripheral blast cells, DCS from CML peripheral blast cells and DFR from KB cells resistant to Methotrexate, but in order to have available a constant source of the enzyme and more homogenous cell population, they will be isolated from CML cells grown in culture employing in essence the similar procedures previously developed. The properties of these enzymes will be compared with those of the analogous enzymes studied by us previously. The interaction of the enzymes with each other and with folate binding macromolecule (FBM) and with various forms of folylpolyglutamate derivatives will then be examined. The analogs to be evaluated for structure activity relationships have been obtained by systematic modification at different positions of the folate molecules. The information thus obtained from these studies will be useful for the design of new analogs with potential selectivity for individual enzymes involved in folate metabolism and thereby, for improving the use of methotrexate in the treatment of cancer.